Purpose:
To investigate the role of Wnt6 in the regulation of human limbal stem/progenitor cells.

Methods:
Stable Wnt6-overexpressing 3T3 cell lines (Wnt6-3T3) were generated using a lentiviral construct pRRL-sin-cPPT-CAG-Wnt6-IRES-GRP. The construct lacking the Wnt6 served as a control. Wnt6-3T3 were sorted into three groups, low-, medium-, and high-Wnt6 according to their GFP expressing level. Freshly isolated limbal epithelial cells (LECs) were cultured on the Wnt6-3T3 feeder cells. The cell proliferation rate, colony forming efficiency (CFE), percentage of p63a-bright cells, and percentage of small cells (diameter≤12mm) were analyzed.

Results:
The GFP expression level correlated with the Wnt6 mRNA level in the Wnt6-3T3 cells by qRT-PCR. The proliferation rate of LECs cultured on Wnt6- 3T3 was reduced by 35% and 24% (p<0.05) in the low- and medium-Wnt6 groups, respectively compared to the control. However, the proliferation rate was increased by 29% in the high -Wnt6 culture. The CFEs on Wnt6-3T3 cultures were reduced by 21% and 14% in low- and medium-Wnt6 groups and increased by 26% (p<0.05) in the high-Wnt6 group compared to controls. LECs cultured on Wnt6-3T3 tended to contain lower percentage (15% lower) of p63a-bright cells in the low-Wnt6 cultures, similar percentage in the medium-Wnt6 culture, and a higher percentage (1.3-fold higher) in the high-Wnt6 culture. Wnt6-3T3 culture tended to generate lower percentage of K12+ cells, especially in the low-Wnt6 group. The amount of small cells was similar in the low-Wnt6 culture and higher in the medium- and high-wnt6 cultures (1.1- and 0.9-fold higher, respectively) compared to the control.

Conclusions:
Different levels of Wnt6 appear to have different effects on human LECs in vitro. High level of Wnt6 supports a faster proliferation rate and better maintains the stem/progenitor phenotype of LECs while low level decreases the proliferation and tends to lose the stem/progenitor phenotype of LECs.<br />